Anti-nucleolin agent-conjugated nanoparticles as radio-sensitizers and mri and/or x-ray contrast agents

ABSTRACT

A composition comprises an anti-nucleolin agent conjugated to nanoparticles, and optionally containing gadolinium. Furthermore, a pharmaceutical composition for treating cancer comprises a composition including an anti-nucleolin agent conjugated to nanoparticles, and a pharmaceutically acceptable carrier. The composition enhances the effectiveness of radiation therapy, enhancing contrast in X-ray imaging techniques, and when gadolinium is present, provide cancer selective MRI contrast agents.

BACKGROUND

Nucleolin [8] is an abundant, non-ribosomal protein of the nucleolus,the site of ribosomal gene transcription and packaging of pre-ribosomalRNA. This 710 amino acid phosphoprotein has a multi-domain structureconsisting of a histone-like N-terminus, a central domain containingfour RNA recognition motifs and a glycine/arginine-rich C-terminus, andhas an apparent molecular weight of 110 kD. While nucleolin is found inevery nucleated cell, the expression of nucleolin on the cell surfacehas been correlated with the presence and aggressiveness of neoplasticcells [3].

The correlation of the presence of cell surface nucleolin withneoplastic cells has been used for methods of determining the neoplasticstate of cells by detecting the presence of nucleolin on the plasmamembranes [3]. This observation has also provided new cancer treatmentstrategies based on administering compounds that specifically targetnucleolin [4].

Nucleic acid aptamers are short synthetic oligonucleotides that foldinto unique three-dimensional structures that can be recognized byspecific target proteins. Thus, their targeting mechanism is similar tomonoclonal antibodies, but they may have substantial advantages overthese, including more rapid clearance in vivo, better tumor penetration,non-immunogenicity, and easier synthesis and storage.

Guanosine-rich oligonucleotides (GROs) designed for triple helixformation are known for binding to nucleolin. This ability to bindnucleolin has been suggested to cause their unexpected ability to effectantiproliferation of cultured prostate carcinoma cells [6]. Theantiproliferative effects are not consistent with a triplex-mediated oran antisense mechanism, and it is apparent that GROs inhibitproliferation by an alternative mode of action. It has been surmisedthat GROs, which display the propensity to form higher order structurescontaining G-quartets, work by an aptamer mechanism that entails bindingto nucleolin due to a shape-specific recognition of the GRO structure;the binding to cell surface nucleolin then induces apoptosis. Theantiproliferative effects of GROs have been demonstrated in cell linesderived from prostate (DU145), breast (MDA-MB-231, MCF-7), or cervical(HeLa) carcinomas and correlates with the ability of GROs to bind cellsurface nucleolin [6].

AS1411, a GRO nucleolin-binding DNA aptamer that has antiproliferativeactivity against cancer cells with little effect on non-malignant cells,was previously developed. AS1411 uptake appears to occur bymacropinocytosis in cancer cells, but by a nonmacropinocytic pathway innonmalignant cells, resulting in the selective killing of cancer cells,without affecting the viability of nonmalignant cells [9]. AS1411 wasthe first anticancer aptamer tested in humans and results from clinicaltrials of AS1411 (including Phase II studies in patients with renal cellcarcinoma or acute myeloid leukemia) indicate promising clinicalactivity with no evidence of serious side effects. Despite a fewdramatic and durable clinical responses, the overall rate of response toAS1411 was low, possibly due to the low potency of AS1411.

Anti-nucleolin agents conjugated to particles, such as aptamersconjugated to gold nanoparticles, have an antiproliferative effect oncancer and tumors. See International Application, InternationalPublication Number WO 2012/167173, entitled “ANTI-NUCLEOLINAGENT-CONJUGATED NANOPARTICLES”, filed 1 Jun. 2012, to Bates et al.Aptamer conjugated gold nanoparticles, in particular, have a similar orgreater antiproliferative effect than the aptamer (anti-nucleolinoligonucleotide) alone, demonstrating similar effects at only 1/10 to1/100 the dosage. Furthermore, these same agents, preferably having afluorescent dye conjugated to the particle or attached to theanti-nucleolin agent, may also be used as imaging agents, both in vivoand ex vivo.

Radiation therapy (RT) has been a mainstay of cancer treatment fordecades and advances in technology mean it is now used to treat morepatients than ever before. The vast majority of cancer patients willreceive RT as part of their treatment, but this therapy is not withoutlimitations or side effects. The ability to deliver sufficient radiationintensity to the tumor without causing unacceptable toxicity in nearbytissues is the overarching problem that constrains the efficacy of RTand leads to local tumor relapse and recurrence.

As the use of RT has grown, the need for potent and selectiveradiosensitizing agents has never been greater. Certain types ofaggressive cancers are particularly difficult to treat with RT. One suchexample is the “triple negative” subtype of breast cancer (TNBC), whichrepresents 15-25% of breast cancer occurrences and is characterized asan aggressive disease with early relapse, low post-recurrence survival,and worse overall survival than other forms of breast cancer. Moreover,TNBC has proven more resistant to radiation therapy and larger doses areneeded for effective treatment. Patients with recurrent TNBC are notablychallenging to treat, as they may already be near their maximum safelifetime radiation dose and there are few clear guidelines for RT inthis setting.

RT also plays an extremely important role in the management of non-smallcell lung cancer (NSCLC) and most patients with NSCLC will receive RT atsome point during their course of treatment. Although RT has provenclinical benefits, it is often inadequate at controlling primary NSCLCtumor growth and preventing recurrence. Increasing the dose of radiationto the tumor is expected to improve effectiveness, but dose escalationis not currently achievable in most cases due to the damage it wouldcause to surrounding healthy tissues.

For many years, there has been interest in developing radiosensitizersto improve the efficacy of RT, but there are currently no agents thatare FDA approved for clinical use specifically for this purpose. Severalchemotherapy drugs have radiosensitizing properties and have been testedconcurrently with RT, but the problem is that they can also make normaltissues more susceptible to radiation damage. Gold nanoparticles (GNP)are well established as radiosensitizers due to their high atomic number(Z), which means they emit secondary radiation when subjected toionizing radiation, plus they are biocompatible. However, the GNPgenerally do not internalize efficiently in cancer cells or accumulatein tumors at sufficiently high concentrations for effectiveradiosensitization, which has limited their clinical utility [26].

Each year in this country, more than 71 million CT scans and 33 millionMRI scans are performed. The purpose for many of these scans is todetect (or rule out) primary or metastatic malignant tumors. Inoncology, medical imaging plays a major role in screening, diagnosis,staging, monitoring therapeutic response, and treatment planning (e.g.for radiation therapy or surgery) for almost all cancer types. In mostof those cases, an intravenous (i.v.) contrast agent is administeredprior to CT or MRI scans to improve visualization of tissues and organs.The short half-life of contrast agents in circulation often requiresrepeated or continuous administration throughout the imaging process.The materials in the contrast agents interact with the imaging modality;for example, iodine in CT contrast absorbs and scatters X-rays, whereasthe gadolinium ions in MRI contrast are paramagnetic and alter theproperties of nearby water molecules. Consequently, contrast agents canenhance differences in signals between adjacent tissues due to theiraltered deposition or interactions, which may depend on tissue type,architecture, or blood flow. Existing CT and MRI contrast agents aresafe in most patients and effectively enhance anatomical imaging, butthey are not disease-specific and largely fail to distinguish malignantfrom benign masses.

Diagnostic imaging is particularly important for lung cancer, which isthe leading cause of cancer deaths in the United States and worldwide.With an aging population of smokers, former smokers and the smallerpopulation of never-smokers who are susceptible to lung adenocarcinomasdue to EGFR mutations, lung cancer is expected to remain a major publichealth problem for many years to come even if smoking cessation effortsare successful. A major factor contributing to the high mortality oflung cancer is the failure to detect lung cancers at an early stagebefore they invade surrounding tissue or spread to other organs (only15% of lung cancers are detected while still localized). The clinicalrelevance of delayed diagnosis in lung cancer is highlighted by the factthat people whose cancer is detected while still localized to theprimary site in the lung have a five year survival rate of 53%, comparedto a 1% chance of surviving five years for those with distant metastasesat the time of diagnosis.

Current lung cancer screening techniques include low-dose helicalcomputed tomography (CT) and single-view posteroanterior chestradiography. Both of these screening techniques have an extremely highfalse positive rate (96.4% for low-dose CT and 94.5% for radiographyaccording to a recent national study). These high rates of falsepositives necessitate additional secondary tests to accurately diagnosethe presence or absence of lung cancer, which reduces thecost-effectiveness of these cancer screening techniques. False positivetest results also cause a significant amount of unnecessary anxiety andemotional stress for patients who believe they have lung cancer.

Another limitation of imaging-based lung cancer screening techniques istheir inability to differentiate between benign and malignant masses.Imaging-based tests identify lung cancer based on the size of nodulespresent in the lungs, with lesions smaller than 4 mm considered benign.Diagnosis based on nodule size fails to identify small malignant lesionsas cancerous and precludes early-stage detection of lung cancer. Inaddition, larger benign growths are wrongly identified as cancerous andrequire more invasive secondary tests to positively diagnose thepresence or absence of lung cancer.

SUMMARY

In a first aspect, the present invention is a composition comprising ananti-nucleolin agent and optionally gadolinium conjugated tonanoparticles.

In a second aspect, the present invention is a pharmaceuticalcomposition for treating cancer, comprising an anti-nucleolin agent andoptionally gadolinium conjugated to nanoparticles and a pharmaceuticallyacceptable carrier.

In a third aspect, the present invention is a pharmaceutical compositionfor enhancing the effectiveness of radiation therapy and/or enhancingcontrast in X-ray imaging techniques, comprising an anti-nucleolin agentconjugated to nanoparticles and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention is a pharmaceuticalcomposition for MRI imaging or enhancing the contrast of an MRI image,comprising an anti-nucleolin agent conjugated to nanoparticles and apharmaceutically acceptable carrier.

In a fifth aspect, the present invention is a method of treating cancer,comprising administering an effective amount of a pharmaceuticalcomposition to a patient in need thereof, followed by radiation therapy.

In a sixth aspect, the present invention is a use of a pharmaceuticalcomposition for the preparation of a medicament for the treatment ofcancer.

In a seventh aspect, the present invention is an agent for MRI imagingand/or enhancing contrast in X-ray imaging techniques comprising acomposition which optionally comprises gadolinium, and apharmaceutically acceptable carrier.

In an eighth aspect, the present invention is a method of imaging cancerby MRI and/or X-ray imaging techniques in vivo, comprising administeringan imaging agent which optionally comprises gadolinium, to a subject;and forming an image of the imaging agent present in the subject by MRIand/or an X-ray imaging technique.

Definitions

The term “conjugated” means “chemically bonded to”.

The term “anti-nucleolin oligonucleotides” refers to an oligonucleotidethat binds to nucleolin.

The term “equivalent aptamer concentration” refers to the concentrationof anti-nucleolin oligonucleotide present in the conjugate.

Tumors and cancers include solid, dysproliferative tissue changes anddiffuse tumors. Examples of tumors and cancers include melanoma,lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breastcancer, prostate cancer, colon cancer, liver cancer, esophageal cancer,brain cancer, lung cancer, ovary cancer, endometrial cancer, bladdercancer, kidney cancer, cervical cancer, hepatoma, and other neoplasms.For more examples of tumors and cancers, see, for example Stedman [1].

“Treating a tumor” or “treating a cancer” means to significantly inhibitgrowth and/or metastasis of the tumor or cancer, and/or killing cancercells. Growth inhibition can be indicated by reduced tumor volume orreduced occurrences of metastasis. Tumor growth can be determined, forexample, by examining the tumor volume via routine procedures (such asobtaining two-dimensional measurements with a dial caliper). Metastasiscan be determined by inspecting for tumor cells in secondary sites orexamining the metastatic potential of biopsied tumor cells in vitro.

A “chemotherapeutic agent” is a chemical compound that can be usedeffectively to treat cancer in humans.

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents which are compatible withpharmaceutical administration. Preferred examples of such carriers ordiluents include water, saline, Ringer's solutions and dextrosesolution. Supplementary active compounds can also be incorporated intothe compositions.

“Medicament,” “therapeutic composition” and “pharmaceutical composition”are used interchangeably to indicate a compound, matter, mixture orpreparation that exerts a therapeutic effect in a subject.

“Antibody” is used in the broadest sense and refers to monoclonalantibodies, polyclonal antibodies, multispecific antibodies, antibodyfragments and chemically modified antibodies, where the chemicalmodification does not substantially interfere with the selectivity andspecificity of the antibody or antibody fragment.

An “anti-nucleolin agent” includes any molecule or compound thatinteracts with nucleolin. Such agents include for example anti-nucleolinantibodies, aptamers such GROs and nucleolin targeting proteins.

“X-ray based imaging techniques” include all imaging techniques whichuse X-rays to form an image, directly or indirectly, including forexample CT scans (also called X-ray computed tomography or computerizedaxial tomography scan (CAT scan)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are images of two MDA231 xenograft mice injectedretro-orbitally with about 200 ng/100 uL of AS1411-Cy5-GNP orCRO-Cy5-GNP. Image was acquired using a PHOTON IMAGER™ after 2 hrs (A)and 6 hrs (B) post injection.

FIGS. 2A, 2B, 2C and 2D are optical micrographs of MCF7 cells treated bysilver enhancement staining for gold nanoparticles, illustratingcomparative accumulation of gold nanoparticles after administration ofthe indicated composition, or without administration.

FIG. 3A is a sketch of different linkers used to conjugate AS1411/CRO togold nanoparticles, and FIG. 3B is a sketch of attaching and detachingaptamers to gold nanoparticles.

FIGS. 4A and 4B show the biodistribution of AS1411-GNP-Cy5: the micewere treated, euthanized and organs were photographed (A) and examinedfor fluorescence (B).

FIG. 5 illustrates the results of uptake studies in MCF-7 cells. BreastCancer Cells (MCF-7) were treated with gold nanoparticles (GNP)conjugated with gadolinium-AS1411 or gadolinium-CRO and a linkedfluorophore (Cy5) for 4 hrs. Confocal microscopy showing the uptake ofthe corresponding Oligo-Gd-GNP-Cy5 conjugate using Cy5 laser excitation(650 nm) and emission (670 nm) in MCF-7 cells.

FIG. 6 illustrates the results of uptake studies in MCF-10A cells.Non-malignant Breast Epithelial Cells (MCF-10A) were treated with goldnanoparticles (GNP) conjugated with gadolinium-AS1411 or gadolinium-CROand a linked fluorophore (Cy5) for 4 hrs. Confocal microscopy showingthe uptake of the corresponding Oligo-Gd-GNP-Cy5 conjugate using Cy5laser excitation (650 nm) and emission (670 nm) in MCF-10A cells.

FIG. 7A illustrates confocal microscopy images of Breast Cancer Cells(MDA-MB-231) treated with gold nanoparticles (GNP) conjugated togadolinium-AS1411 (GNP-Gd-AS1411) or AS1411 (GNP-AS1411) for 4 hrs, andtreated with 100 cGy X-ray. DNA damage response marker phospho-yH2AX(red) foci was detected in the damage nuclei (blue) of MDA-MB-231 cells.

FIG. 7B is a graph illustrating the average foci per nuclei for BreastCancer Cells (MDA-MB-231) treated with gold nanoparticles (GNP)conjugated to gadolinium-AS1411 (GNP-Gd-AS1411) or AS1411 (GNP-AS1411)for 4 hrs, and treated with 100 cGy X-ray, with bars indicating thestandard deviation (SD).

FIG. 8 illustrates Breast Cancer Cells (MDA-MB-231) plated in 35 mmdishes and treated with gold nanoparticle conjugated to gadolinium andAS1411 (0.03 mg/ml gold concentration). After 4 hrs dishes were radiatedusing X-Rad160/225 radiator at 100 cGy. Dishes were further incubatedfor 10 days. After incubation the colonies fix with 4% paraformaldehydein phosphate buffer saline (PBS) and stained with 0.4% crystal violet.

FIG. 9A illustrates the results of relaxivity analysis at 9.4 T for Goldnanoparticles coated with AS1411 and Gd(III)-DO3A-SH. FIG. 9Billustrates the results of relaxivity analysis at 9.4 T for Goldnanoparticles coated with CRO (control) and Gd(III)-DO3A-SH. FIG. 9Cillustrates the results of relaxivity analysis at 9.4 T for goldnanoparticles coated with Gd(III)-DO3A-SH. FIG. 9D illustrates theresults of relaxivity analysis at 9.4 T for MULTIHANCE® (gadobenatedimeglumine). FIG. 9E illustrates a Gold Nanoparticle coated with 24Gd(III)-DO3A-SH and 13 AS1411/CRO.

FIG. 10A illustrates X-ray attenuation intensities in Hounsfield Unit(HU) as a function of GNP concentrations for GNS/Gd(III)-DOTA-SH/AS1411construct (green), GNP/AS1411 (red), and Iopamidol (blue).

FIG. 10B illustrates a table of X-ray attenuation intensities inHounsfield Unit (HU) for various constructs.

FIG. 11A illustrates lung cancer cells treated with gold nanoparticlesand gold nanoparticle-aptamer conjugates without exposure to X-rayradiation.

FIG. 11B illustrates treated lung cancer cells with exposure to X-rayradiation.

FIG. 11C is a graph illustrating the percent change in absorbance of thecells with and without exposure to X-ray radiation.

FIG. 12 illustrates confocal microscopy images of breast cancer cellstreated with gold nanoparticles conjugated to AS1411 (GNP-AS1411).

FIG. 13A illustrates the breast cancer cell survival fraction aftertreatment with 1.38 μg/mL gold nanoparticles and goldnanoparticle-aptamer conjugates.

FIG. 13B illustrates the cancer cell survival fraction after treatmentwith 2.76 μg/mL gold nanoparticles and gold nanoparticle-aptamerconjugates.

FIG. 13C illustrates the cancer cell survival fraction after treatmentwith 5.5 μg/mL gold nanoparticles and gold nanoparticle-aptamerconjugates.

FIG. 14A illustrates breast cancer cells treated with gold nanoparticlesand gold nanoparticle-aptamer conjugates.

FIG. 14B is a graph illustrating the survival fraction of the cells.

FIG. 15A illustrates uptake of fluorescent-labeled goldnanoparticle-gadolinium-oligomer conjugates in non-malignant breastcells.

FIG. 15B illustrates uptake and selective retention offluorescent-labeled gold nanoparticle-gadolinium-oligomer conjugates inmalignant breast cancer cells.

FIG. 16A illustrates uptake of fluorescent-labeled goldnanoparticle-gadolinium-oligomer conjugates in non-malignant lung cells.

FIG. 16B illustrates uptake and selective retention offluorescent-labeled gold nanoparticle-gadolinium-oligomer conjugates inmalignant lung cancer cells.

FIG. 17A illustrates the change in relaxivity of goldnanoparticle-oligomer-gadolinium conjugates in water.

FIG. 17B illustrates the change in relaxivity of goldnanoparticle-oligomer-gadolinium conjugates in cells after 48 hours oftreatment.

FIG. 17C illustrates the change in relaxivity of goldnanoparticle-oligomer-gadolinium conjugates in cells after 96 hours oftreatment.

FIG. 18 illustrates the percent contrast enhancement for goldnanoparticle-oligomer conjugates and a commercially-available MRIcontrast agent.

DETAILED DESCRIPTION

The present invention includes anti-nucleolin agents conjugated toparticles comprising metals, such as aptamers conjugated to goldnanoparticles, that are effective radio-sensitizers for treating cancer.The nanoparticles are selectively taken-up by cancer cells and enhancethe effects of RT on those cells. This enhances the effectiveness of RT,and/or allows a low effective dose of radiation to be used during RT.Furthermore, the nanoparticles may optionally also contain gadolinium,for example10-(2-sulfanylethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid(H3-DO3A-SH) coordinated to a trivalent gadolinium ion (Gd-DO3A-SH),which is then bound to the gold nanoparticles through the sulfur atom ina manner similar to thiolated-AS1411, resulting in a mono-layer coatingthat is a mixture of AS1411 and Gd-DO3A-SH on the gold nanoparticles.The Gd ions of Gd-DO3A-SH enhance the relaxivity (speed up therelaxation rate) of nearby water molecules during an MRI scan andcontribute to an increase in contrast when present in the sample beingscanned. In addition, both the gold nanoparticles and the Gd ionsenhance the absorption and scattering of X-rays, and increase thecontrast when present in the sample being scanned or imaged using X-raybased imaging techniques, such as CT scanning. The combination ofanti-nucleolin agents (cancer targeting) and gadolinium (MRI contrastenhancement) combine to result in a cancer-targeting MRI-contrast agent.Furthermore the combination of anti-nucleolin agents (cancer targeting)and gold nanoparticle (X-ray contrast enhancement) and optionallygadolinium (MRI contrast enhancement and X-ray contrast enhancement)combine to result in a cancer-targeting MRI-contrast and X-ray (CT scan)contrast agent.

There are several unmet needs that can be addressed by thesecancer-targeted contrast agent, namely: (1) better specificity todifferentiate between cancerous and non-cancerous lesions and reducefalse positives that can lead to over-treatment, (2) improvedsensitivity so that they could be used as screening tools for earlydetection, and (3) reduced toxicity to allow use in patients withcompromised renal function for whom existing contrast agents arecontraindicated. These advantages are especially useful in lung cancerscreening, particularly early detection of lung cancer.

In addition, anti-nucleolin agent-conjugated nanoparticles have a longerhalf-life in circulation than currently available contrast agents. Thelonger half-life in circulation eliminates the need for continuousintravenous administration during imaging, which greatly improvespatient comfort. Anti-nucleolin agent-conjugated nanoparticle contrastagents may be administered multiple days before an imaging scan isperformed.

Anti-nucleolin agents include (i) aptamers, such as GROs; (ii)anti-nucleolin antibodies; and (iii) nucleolin targeting proteins.Examples of aptamers include guanosine-rich oligonucleotides (GROs).Examples of suitable oligonucleotides and assays are also given inMiller et al. [7]. Characteristics of GROs include:

(1) having at least 1 GGT motif,

(2) preferably having 4-100 nucleotides, although GROs having many morenucleotides are possible,

(3) optionally having chemical modifications to improve stability.

Especially useful GROs form G-quartet structures, as indicated by areversible thermal denaturation/renaturation profile at 295 nm [6].Preferred GROs also compete with a telomere oligonucleotide for bindingto a target cellular protein in an electrophoretic mobility shift assay[6]. In some cases, incorporating the GRO nucleotides into largernucleic acid sequences may be advantageous; for example, to facilitatebinding of a GRO nucleic acid to a substrate without denaturing thenucleolin-binding site. Examples of oligonucleotides are shown in Table1; preferred oligonucleotides include SEQ IDs NOs: 1-7; 9-16; 19-30 and31 from Table 1.

TABLE 1  Non-antisense GROs that bind nucleolin and non-binding controls^(1,2,3). SEQ ID GRO Sequence NO: GRO29A¹tttggtggtg gtggttgtgg tggtggtgg 1 GRO29-2tttggtggtg gtggttttgg tggtggtgg 2 GRO29-3tttggtggtg gtggtggtgg tggtggtgg 3 GRO29-5tttggtggtg gtggtttggg tggtggtgg 4 GRO29-13 tggtggtggt ggt 5 GRO14Cggtggttgtg gtgg 6 GRO15A gttgtttggg gtggt 7 GRO15B² ttgggggggg tgggt 8GRO25A ggttggggtg ggtggggtgg gtggg 9 GRO26B¹ggtggtggtg gttgtggtgg tggtgg 10 GRO28A tttggtggtg gtggttgtgg tggtggtg 11GRO28B tttggtggtg gtggtgtggt ggtggtgg 12 GRO29-6ggtggtggtg gttgtggtgg tggtggttt 13 GRO32Aggtggttgtg gtggttgtgg tggttgtggt gg 14 GRO32Btttggtggtg gtggttgtgg tggtggtggt tt 15 GRO56Aggtggtggtg gttgtggtgg tggtggttgt 16 ggtggtggtg gttgtggtgg tggtgg CROtttcctcctc ctccttctcc tcctcctcc 18 GRO A ttagggttag ggttagggtt aggg 19GRO B ggtggtggtg g 20 GRO C ggtggttgtg gtgg 21 GRO D ggttggtgtg gttgg 22GRO E gggttttggg 23 GRO F ggttttggtt ttggttttgg 24 GRO G¹ggttggtgtg gttgg 25 GRO H¹ ggggttttgg gg 26 GRO I¹ gggttttggg 27 GRO J¹ggggttttgg ggttttgggg ttttgggg 28 GRO K¹ ttggggttgg ggttggggtt gggg 29GRO L¹ gggtgggtgg gtgggt 30 GRO M¹ ggttttggtt ttggttttgg ttttgg 31GRO N² tttcctcctc ctccttctcc tcctcctcc 32 GRO O²cctcctcctc cttctcctcc tcctcc 33 GRO P² tggggt 34 GRO Q² gcatgct 35GRO R² gcggtttgcg g 36 GRO S² tagg 37 GRO T² ggggttgggg tgtggggttg ggg38 ¹Indicates a good plasma membrane nucleolin-binding GRO. ²Indicates anucleolin control(non-plasma membrane nucleolin binding). ³GRO sequencewithout ¹ or ² designations have some anti-proliferative activity.

Any antibody that binds nucleolin may also be used. In certaininstances, monoclonal antibodies are preferred as they bind single,specific and defined epitopes. In other instances, however, polyclonalantibodies capable of interacting with more than one epitope onnucleolin may be used. Many anti-nucleolin antibodies are commerciallyavailable, and are otherwise easily made. See, for example, US PatentApplication Publication No. US 2013/0115674 to Sutkowski et al. Table 2lists a few commercially available anti-nucleolin antibodies.

TABLE 2 commercially available anti-nucleolin antibodies AntigenAntibody Source source p7-1A4 Mouse monoclonal Developmental StudiesXenopus laevis antibody (mAb) Hybridoma Bank oocytes Sc-8031 mouse mAbSanta Cruz Biotech human Sc-9893 goat polyclonal Santa Cruz Biotechhuman Ab (pAb) Sc-9892 goat pAb Santa Cruz Biotech human Clone 4E2 mousemAb MBL International human Clone 3G4B2 mouse mAb Upstate Biotechnologydog (MDCK cells) Nucleolin, Human MyBioSource human (mouse mAb) Purifiedanti-Nucleolin- BioLegend human Phospho, Thr76/Thr84 (mouse mAb) RabbitPolyclonal Novus Biologicals human Nucleolin Antibody Nucleolin (NCL,C23, US Biological human FLJ45706, FLJ59041, Protein C23) Mab Mo xHuNucleolin (NCL, Nucl, C23, US Biological human FLJ45706, Protein C23)Pab Rb xHu Mouse Anti-Human Cell Sciences human Nucleolin Phospho-Thr76/Thr84 Clone 10C7 mAb Anti-NCL/Nucleolin (pAb) LifeSpan Bioscienceshuman NCL purified MaxPab mouse Abnova human polyclonal antibody (B02P)NCL purified MaxPab rabbit Abnova human polyclonal antibody (D01P) NCLmonoclonal antibody, Abnova human clone 10C7 (mouse mAb) NucleolinMonoclonal Enzo Life Sciences human Antibody (4E2) (mouse mAb)Nucleolin, Mouse Monoclonal Life Technologies human Antibody CorporationNCL Antibody (Center E443) Abgent human (rabbit pAb) Anti-Nucleolin,clone 3G4B2 EMD Millipore human (mouse mAb) NCL (rabbit pAb) ProteintechGroup human Mouse Anti-Nucleolin Active Motif human Monoclonal Antibody,Unconjugated, Clone 3G4B20 Nsr1p - mouse monoclonal EnCor Biotechnologyhuman Nucleolin (mouse mAb) Thermo Scientific human Pierce ProductsNucleolin [4E2] antibody GeneTex human (mouse mAb)

Nucleolin targeting proteins are proteins, other than antibodies, thatspecifically and selectively bind nucleolin. Examples include ribosomalprotein S3, tumor-homing F3 peptides [26, 27] and myosin H9 (anon-muscle myosin that binds cell surface nucleolin of endothelial cellsin angiogenic vessels during tumorigenesis).

Anti-nucleolin agents may be conjugated to particles made of a varietyof materials solid materials, including (1) metals and high molecularweigh elements; and (2) metal oxides. Metals and elements, preferablynon-magnetic metals and elements, include gold, silver, palladium,iridium, platinum and alloys thereof. Oxides include zirconium dioxide,palladium oxide, barium sulfate, thorium oxide, uranium oxide andcomplex oxides thereof, such as barium titanate. Preferably, theparticles are non-toxic. The particles are preferably nanoparticleshaving an average particle diameter of 1-100 nm, more preferably 1-50nm, including 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90 and 95 nm.

Oligonucleotides and proteins have been attached to solid materials,such metals and elements, oxides, semiconductors and polymers, by avariety of techniques. These same techniques may be used to attachanti-nucleolin agents to particles. Further attachment of gadoliniumcomplexes to the anti-nucleolin agent conjugated nanoparticles(conjugates), allows the conjugates to be used as MRI contrast agents,both in vivo and ex vivo.

Anti-nucleolin agent-conjugated nanoparticles may be used to formulate apharmaceutical composition for radio-sensitizers for treating cancer andtumors by RT, and targeting cancer cells expressing cell surfacenucleolin, by forming mixtures of the anti-nucleolin agent conjugatednanoparticles and a pharmaceutically acceptable carrier, such as apharmaceutical composition. Methods of treating cancer in a subjectinclude administering a therapeutically effective amount of ananti-nucleolin agent conjugated nanoparticles followed by RT. The smallsize of nanoparticles allows nanoparticle conjugates to cross theblood-brain barrier, which enables imaging and treatment of braintumors.

Particularly preferred compositions are aptamers conjugated to goldnanoparticles, and optionally further conjugated to gadoliniumcomplexes. Gold nanoparticles (GNPs) exhibit low toxicity, versatilesurface chemistry, light absorbing/scattering properties, and tunablesize. Aptamers effectively cap gold particles and prevent aggregation,are safe, stable, easy to synthesize, and non-immunogenic. Aptamerconjugated GNPs offer improved efficacy of RT in vivo. Aptamerconjugated GNP are highly selective for cancer cells over normal cells,and when attached to cyanine dyes are excellent imaging agents, forexample Cy2, Cy3, Cy5, Cy®5.5, Cy7, Alexa Fluor® 680, Alexa Fluor 750,IRDye® 680, and IRDye® 800CW (LI-COR Biosciences, Lincoln, Nebr.); andwhen attached to gadolinium complexes also act as MRI contrast agentsspecific for cancer cells. Aptamer conjugated GNP, and optionallyattached to gadolinium complexes may be used as an imaging agent, MRIcontrast agents, and may be administered as compositions which furthercontain a pharmaceutically acceptable carrier. The imaging agent may beadministered to a subject in a method of imaging cancer in vivo, to forman image of the imaging agent present in the subject, by MRI.

The amounts and ratios of compositions described herein are all byweight, unless otherwise stated. Accordingly, the number ofanti-nucleolin agents per nanoparticle may vary when the weight of thenanoparticle varies, even when the equivalent anti-nucleolin agentconcentration (or equivalent aptamer concentration) is otherwise thesame. For example, the number of anti-nucleolin agent molecules pernanoparticle may vary from 2 to 10,000, or 10 to 1000, including 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 and 900.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration, including intravenous, intradermal,subcutaneous, oral, inhalation, transdermal, transmucosal, and rectaladministration. Solutions and suspensions used for parenteral,intradermal or subcutaneous application can include a sterile diluent,such as water for injection, saline solution, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; buffers such as acetates, citrates orphosphates, and agents for the adjustment of tonicity such as sodiumchloride or dextrose. The pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid so as to be administered using a syringe.Such compositions should be stable during manufacture and storage andare preferably preserved against contamination from microorganisms suchas bacteria and fungi. The carrier can be a dispersion mediumcontaining, for example, water, polyol (such as glycerol, propyleneglycol, and liquid polyethylene glycol), and other compatible, suitablemixtures. Various antibacterial and anti-fungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, cancontain microorganism contamination. Isotonic agents such as sugars,polyalcohols, such as mannitol, sorbitol, and sodium chloride can beincluded in the composition. Compositions that can delay absorptioninclude agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activeagents, and other therapeutic components, in the required amount in anappropriate solvent with one or a combination of ingredients asrequired, followed by sterilization. Methods of preparation of sterilesolids for the preparation of sterile injectable solutions includevacuum drying and freeze-drying to yield a solid.

Radiation treatment (RT) may be by any form of radiation, such as X-rays(for example megavolt energy X-rays), Brachy therapy, proton radiation,and neutron radiation. Also possible is to use doses and/or energy of RTthat would normally be considered subclinical; because theanti-nucleolin agent-conjugated nanoparticles enhance the effectivenessof RT, the dosages are effective to kill or reduce the growth of cancercells and tumors.

Anti-nucleolin agent-conjugated nanoparticles which contain gadoliniumare effective MRI contrast agents, and may also be used to image cancercells, including individual cancer cells. For example, theanti-nucleolin agent-conjugated nanoparticles which contain gadoliniummay be administered to a patient to determine if cancer cells arepresent in lymph nodes, thus avoiding the removal of lymph node for thesole purpose of determining if they contain cancer cells. Another usecan be to avoid the need for a biopsy. The anti-nucleolinagent-conjugated nanoparticles which contain gadolinium may beadministered to a patient to determine if cancer is preseht in a lump,has metastasized to other location in the body, or to determine if allcancer from a tumor has been removed during surgery.

Anti-nucleolin agent-conjugated nanoparticles which optionally containgadolinium are effective X-ray contrast agents, and may also be used toimage cancer cells, including individual cancer cells. For example, theanti-nucleolin agent-conjugated nanoparticles which optionally containgadolinium may be administered to a patient to determine if cancer cellsare present in lymph nodes, thus avoiding the removal of lymph node forthe sole purpose of determining if they contain cancer cells. Anotheruse can be to avoid the need for a biopsy. The anti-nucleolinagent-conjugated nanoparticles which optionally contain gadolinium maybe administered to a patient to determine if cancer is present in alump, has metastasized to other location in the body, or to determine ifall cancer from a tumor has been removed during surgery.

The pharmaceutical composition described herein may further compriseother therapeutically active compounds, and/or may be used inconjunction with physical techniques as noted herein which are suitablefor the treatment of cancers and tumors. Examples of commonly usedtherapeutically active compounds include vinorelbine (Navelbine®),mitomycin, camptothecin, cyclophosphamide (Cytoxin®), methotrexate,tamoxifen citrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide,paclitaxel (Taxol®), docetaxel, vinblastine, imatinib mesylate(Gleevec®), anthracycline, letrozole, arsenic trioxide (Trisenox®),anastrozole, triptorelin pamoate, ozogamicin, irinotecan hydrochloride(Camptosar®), BCG live (Pacis®), leuprolide acetate implant (Viadur),bexarotene (Targretin®), exemestane (Aromasin®), topotecan hydrochloride(Hycamtin®), gemcitabine HCL(Gemzar®), daunorubicin hydrochloride(Daunorubicin HCL®), toremifene citrate (Fareston), carboplatin(Paraplatin®), cisplatin (Platinol® and Platinol-AQ®) oxaliplatin andany other platinum-containing oncology drug, trastuzumab (Herceptin®),lapatinib (Tykerb®), gefitinib (Iressa®), cetuximab (Erbitux®),panitumumab (Vectibix®), temsirolimus (Torisel®), everolimus(Afinitor®), vandetanib (Zactima™), vemurafenib (ZelborafrM), crizotinib(Xalkori®), vorinostat (Zolinza®), bevacizumab (Avastin®), hyperthermia,gene therapy and photodynamic therapy.

In the treatment of cancer, an appropriate dosage level of thetherapeutic agent will generally be about 0.01 to 500 mg per kg patientbody weight per day which can be administered in single or multipledoses. Preferably, the dosage level will be about 0.1 to about 250 mg/kgper day; more preferably about 0.5 to about 100 mg/kg per day. Asuitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within thisrange the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day.The compounds may be administered on a regimen of 1 to 4 times per day,preferably once per day prior to RT. Administration by continuousinfusion is also possible. All amounts and concentrations ofanti-nucleolin oligonucleotide conjugated gold nanoparticles are basedon the amount or concentration of anti-nucleolin oligonucleotide only.

Pharmaceutical preparation may be pre-packaged in ready-to-administerform, in amounts that correspond with a single dosage, appropriate for asingle administration referred to as unit dosage form. Unit dosage formscan be enclosed in ampoules, disposable syringes or vials made of glassor plastic.

However, the specific dose level and frequency of dosage for anyparticular patient may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the patient undergoing therapy.

EXAMPLES

AS1411-linked gold nanoparticles for treating cancer and for cancerimaging were synthesized. Studies to assess the anticancer activity ofAS1411 linked to 5 nm gold nanoparticles indicate that the conjugateshave greatly enhanced antiproliferative effects on breast cancer cellscompared to AS1411 (SEQ ID NO. 10) alone. Microscopic examinationrevealed increased uptake in breast cancer cells for GNP-AS1411 comparedto GNP alone or GNP conjugated to a control oligonucleotide. Inaddition, GNP-AS1411 induced breast cancer cell vacuolization and death,similar to that seen at higher concentrations of AS1411. The GI50 valuesfor AS1411 conjugated GNP against breast cancer cells are in the 50-250nM range, compared to 1-10 uM range for unconjugated AS1411 (equivalentaptamer concentration). Studies indicate that these AS1411-GNPs haveselective accumulation in tumor tissue following systemic administrationin mice. Moreover, AS1411-GNPs retained the cancer-selectivity of AS1411and had no effect on non-malignant cells.

Preparation of Aptamer Conjugated Gold Nanoparticles (GNP)

The aptamers AS1411 and CRO (the control oligonucleotide) with 5′ primethiol modification and or 3′ fluorophore Cy5 were purchased fromIntegrated DNA Technologies (IDT).

AS1411 with thiol link at 5′: 5′-/5ThioMC6-D/TTT TTT GGT GGT GGTGGT TGT GGT GGT GGT GGT TT/-3′. CRO with thiol link at 5′:5′-/5ThioMC6-D/TTT TTT CCT CCT CCT CCT TCT CCT CCT CCT CCT TT/-3′.AS1411 with thiol link at 5′ and fluorophore Cy5 at 3′:5′-/5ThioMC6-D/TTT TTT GGT GGT GGT GGT TGT GGT GGT GGT GGT TT/Cy5Sp/-3′.CRO with thiol link at 5′ fluorophore Cy5 at 3′:5′-/5ThioMC6-D/TTT TTT CCT CCT CCT CCT TCT CCT CCT CCT CCT TT/Cy5Sp/-3′.

The thiol ends of aptamers were reduced by tri(2-carboxyethyl) phosphineTECP (50 mM) which is active in slightly acidic pH 6.5 of Tris-EDTA (10mM) solution for 4-8 hours at room temperature. The solution of aptamersand TECP was purified using NAP-columns sephadex G-25. AccurateSpherical Gold nanoparticles 5 nm was purchased from NANOPARTZ and/orTED PELLA INC. The gold nanoparticles were filtered using 0.5 micronsyringe filter. Gold nanoparticles and aptamers were mixed in the molarratio of 1:40 in 25 ml RNAse and DNAse free water at room temperatureovernight. Excess reagents were then removed by centrifugation at 15000rpm for 20 min, followed by 3× wash with RNAse and DNAse free water andcentrifugation to remove any unbound aptamers. To quantify the amount ofaptamers conjugated on the nanoparticles surface, the aptamer conjugatedGNP was incubated in 0.1M DTT at room temperature followed by theseparation from the GNP by centrifugation. The supernatant was dilutedand measured either spectrophotometically (A260 nm), then calculatingthe concentration from the aptamers standard dilution curve or byNanoDrop 2000 UV-VIS spectrophotometer. Similarly, the concentration ofgold nanoparticles was calculated using spectrophotometric opticaldensity (OD) at 511 nm and plotting the standard dilution curve toextrapolate the concentration of gold nanoparticles and the standarddata provided by vendors.

FIGS. 1A and 1B are images of two MDA231 xenograft mice (nude miceinjected with MDA231 cancer cells), 2 hours (A) and 6 hours (B) afterinjection retro-orbitally with AS1411 conjugated with a fluorophor(cyanine dye Cy5) and gold nanoparticles (AS1411-Cy5-GNP), or with acontrol oligonucleotide conjugated with the fluorophor (cyanine dye Cy5)and gold nanoparticles (CRO-Cy5-GNP). The images were acquired using aPHOTON IMAGER™ at 680 nm. The images show that the tumors accumulateAS1411-Cy5-GNP, while CRO-Cy5-GNP is not accumulated.

FIGS. 2A-D are optical micrographs of MCF7 cells treated by silverenhancement staining for gold nanoparticles, illustrating comparativeaccumulation of gold nanoparticles after administration of the indicatedcomposition, or without administration. As shown, no significantaccumulation occurs after administration of unconjugated goldnanoparticles (FIG. 2B), and only slight accumulation occurs afteradministration of control oligonucleotide-conjugated gold particles(FIG. 2C). Administration of AS1411-conjugated gold nanoparticles,however, results in significant accumulation of gold nanoparticles, asindicated by the arrows (FIG. 2D). Non-treated cells are also shown(FIG. 2A).

Comparison of Different Routes of Injection for Delivery of AS1411-GNPto Target Tissue

Three different routes of injection for delivery of GNP-AS1411 to targettissue were tested: intraperitoneal, intravenous, via tail vein,retro-orbital, injection. Based on pilot studies, it was determined thatfor long term and repeated injections (as in therapeutic dosing),intraperitoneal injection was preferred for its convenience and becausethe slower biodistribution (compared to intravenous or retro-orbital)was not a concern. For imaging, either tail vein or retro-orbitalinjections were used because it delivered the drug directly into theblood, resulting in more rapid systemic distribution and avoidingresidual signal in the peritoneum that was observed when deliveringthrough the intraperitoneal route.

Effect of GNP Size and Linkers Length on Cell Proliferation

Syntheses and analyses of GNPs and linkers were performed as follows:colloid spherical gold nanoparticles of different size (5, 10, 15 nm)were purchased from Ted Pella Inc. (Redding, Calif.) and Nanopartz(Loveland, Colo.). Size analyses of these gold nanoparticles wereconfirmed using PARTICLES SIZE ANALYZER 90 PLUS (Brookhaven Instrument),and the sizes of gold nanoparticles were within the ranges as describedby the manufacturers. Fluorophore (Cy5)-linked oligonucleotides (AS1411and CRO), with or without carbon spacers and thiol groups, werepurchased from Integrated DNA Technologies (San Diego, Calif.). Cy5, orcyanine-5 phosphoramidite(1-[3-(4-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidityl]propyl]-3,3,3′,3′-tetramethylindodicarbocyaninechloride) has the structure shown in Formula I:

The linkers, C3-thiol(1-O-dimethoxytrityl-propyl-disulfide,1′-succinyl-lcaa), MC6-D/iSP-9(9-O-dimethoxytrityl-triethyleneglycol,1[(2-cyanoethyl)-(N,N-diisopropyl)]), andMC6-D/iSP-18(18-O-dimethoxytrityl hexaethyleneglycol,1[(2-cyanoethyl)-(N,N-diisopropyl)]), have the structures shownin Formulas II, III and IV, respectively:

FIG. 3A is a cartoon representation of the various agents tested. FIG.3B is an illustration of the reaction to form conjugates, and toseparate the parts of the conjugates.

In Vivo Biodistribution of AS1411-GNP Conjugated to Fluorophore Cy5

The use of multimodal imaging approaches utilizing optical and microCTwas useful for detection of primary or disseminated breast cancertumors. In this experiment a Cy5 fluorophore was linked to the 5′-end ofAS1411 and conjugated to the GNP (to give GNP-AS1411-Cy5), in order toevaluate its utility as a complex not only for optical imaging but alsoas a contrast agent for computed tomography (CT). A similar constructwith CRO was synthesized as a control. Nude mice with MDA-MB-231 breastcancer xenografts on each flank were administered a single injection offluorophore-oligonucleotide-GNP. Images were acquired using IVIS ImagingSystem/MAESTRO Fluorescence Imaging and preliminary data showed thatGNP-AS1411-Cy5 (1 mg/kg) concentration in the tumor is many times morethan that using AS1411-Cy5 without GNP (10 mg/kg), or GNP—CRO-Cy5. Itwas noted that all mice exhibited strong signals on their extremities(legs and paws) and tails; these were artifacts from the urine and fecesof the mice in cage where they were housed (possibly due to afluorescent substance in the animal feed). Washing the mice and housingthem in new clean new cages before imaging can prevent this problem.Biodistribution analysis also confirmed that, besides liver, kidney andintestine, most of the GNP-AS1411 accumulated in the tumor (FIG. 4).This is a proof of concept that conjugating AS1411 to gold nanoparticlescan specifically target the tumors. Mice were treated by intraperitonealinjection with the indicated substances and were imaged after 96 h.Images showed high accumulation of GNP-AS1411-Cy5 (1 mg/kg aptamerconcentration) in breast cancer xenograft, as compared to AS1411 (10mg/Kg) and GNP-CRO alone.

Uptake Studies in MCF-7 Cells

Breast Cancer Cells (MCF-7) were treated with gold nanoparticles (GNP)conjugated with gadolinium-AS1411 or gadolinium-CRO and a linkedfluorophore (Cy5) for 4 hrs. Confocal microscopy showing the uptake ofthe corresponding Oligo-Gd-GNP-Cy5 conjugate using Cy5 laser excitation(650 nm) and emission (670 nm) in MCF-7 cells. The results are shown inFIG. 5.

Uptake Studies in MCF-10A Cells

Non-malignant Breast Epithelial Cells (MCF-10A) were treated with goldnanoparticles (GNP) conjugated with gadolinium-AS1411 or gadolinium-CROand a linked fluorophore (Cy5) for 4 hrs. Confocal microscopy showingthe uptake of the corresponding Oligo-Gd-GNP-Cy5 conjugate using Cy5laser excitation (650 nm) and emission (670 nm) in MCF-10A cells. Theresults are shown in FIG. 6.

DNA Damage Response

Confocal Microscopy images are shown in FIG. 7. (A). Breast Cancer Cells(MDA-MB-231) were treated with gold nanoparticles (GNP) conjugated togadolinium-AS1411 (GNP-Gd-AS1411) or AS1411 (GNP-AS1411) for 4 hrs, andtreated with 100 cGy X-ray. DNA damage response marker phospho-yH2AX(red) foci was detected in the damage nuclei (blue) of MDA-MB-231 cells(8). Graph showing the average foci per nuclei, bars indicates standarddeviation (SD).

Confocal microscopy images are shown in FIG. 12. Breast Cancer Cells

(MDA-MB-231) were treated with 1.38 μg/mL gold nanoparticles (GNP)conjugated to AS1411 (GNP-AS1411) for 4 hrs, and treated with 100 cGyX-ray. DNA damage response marker phospho-yH2AX (Bethyl Laboratory)(red) foci was detected in the damage nuclei (blue) of MDA-MB-231 cells.A UV treated+ve control is also shown.

Clonogenic Assay of Breast Cancer Cells

Breast Cancer Cells (MDA-MB-231) were plated in 35 mm dishes and treatedwith gold nanoparticle conjugated to gadolinium and AS1411 (0.03 mg/mlgold concentration). After 4 hrs dishes were radiated using X-Rad160/225radiator at 100 cGy. Dishes were further incubated for 10 days. Afterincubation the colonies fix with 4% paraformaldehyde in phosphate buffersaline (PBS) and stained with 0.4% crystal violet. The results are shownin FIG. 8.

Relaxivity Analysis

FIG. 9 shows the results of the relaxivity analysis at 9.4 T for: (9A)Gold nanoparticles coated with AS1411 and Gd(III)-DO3A-SH (9B) Goldnanoparticles coated with CRO (control) and Gd(III)-DO3A-SH (9C) Goldnanoparticles coated with Gd(III)-DO3A-SH and (9D) MULTIHANCE®(gadobenate dimeglumine). FIG. 9E illustrates a Gold Nanoparticle coatedwith 24 Gd(III)-DO3A-SH and 13 AS1411/CRO.

Gadolinium-Functionalized Gold Nanoparticle CT/MRI Contrast Agent withCancer Targeting Capabilities

Gold nanoparticles (GNP) provide contrast in computed tomography (CT)images and other X-ray based imaging techniques due to their high atomicmass, and may be modified with bioactive coatings to increase theirfunctionality. We have tailored spherical GNPs (˜4 nm) with a T1gadolinium-based magnetic resonance imaging (MRI) contrast agent(Gd(III)-DO3A-SH) and therapeutic/cancer-targeting DNA aptamer (AS1411)for cancer imaging and therapy. GNP coated with Gd(III)-DO3A-SH andAS1411 or CRO had hydrodynamic diameters of 13.45±2.11 and 19.01±2.51nm, respectively, and zeta potentials of −13.83±0.74 and −52.62±1.01 mV.Both solutions were stable for more than 6 months in physiologicalbuffer solutions. EDAX analysis of GNP—Gd(III)-DO3A-AS1411 and GNPGd(III)-DO3A-CRO yielded 28±5 and 23±4 Gd centers per GNP, respectively,compared to 15±1 Gd centers per GNP for GNP—Gd(III)-DO3A solutions.AS1411 was detected on the gold nanoparticle surface using Quant-iT™OliGreen® ssDNA reagent via fluorescence imaging studies on purifiedsamples of GNP Gd(III)-DO3A-AS1411, GNP—Gd(III)-DO3A and GNP-AS1411.

The GNP-Gd(III)-DO3A-AS1411/CRO probes have been assessed for theirefficacy as CT and/or MRI contrast agents. At both 9.4 and 3.0 Tesla,solutions of GNP-Gd(III)-DO3A-SH-AS1411 and GNP-Gd(III)-DO3A-SH-CROgenerate higher relaxivity than GNP-Gd(III)-DO3A, industry standardMULTIHANCE® (gadobenate dimeglumine) or Gd(III)-DO3A-SH, Table 3. In CTscans, solutions of GNP-Gd(III)-DO3A-AS1411 and GNP-Gd(III)-DO3A-CROyield significantly higher X-Ray attenuation (Hounsefield unit permilligram milliliter) values in comparison to Iopamidol,GNP-Gd(III)-DO3A and citrate capped gold, Table 3.

TABLE 3 Relaxivity and X-ray attenuation data Relaxivity RelaxivityHounsefield (mM⁻¹s⁻¹) (mM⁻¹s⁻¹) Unit (HU) Samples at 9.4 T at 3.0 T mg⁻¹ml GNP-(Gd(III) DO3A SH)-AS1411 24.83 27.51 227.46 GNP-(Gd(III) DO3ASH)-CRO 15.67 31.61 250.66 GNP-(Gd(III) DO3A SH) 5.56 — 81.53 Gd(III)DO3A SH 2.29 — MULTIHANCE ® (gadobenate 3.75 — — dimeglumine) GNP AS1411— — 83.03 GNP CRO — — 113.47 GNP Citrate Capped — — 92.47 Iopamidol — —71.07

CT Contrast

The Hounsfield unit is a normalized index of x-ray attenuation rangingfrom −1000 (air) to +1000 (bone) with water being 0 and is used in CTimaging to evaluate contrast. We have performed 5 microCT studies usinga MicroCAT-II (Siemens, Knoxville, Tenn.) to evaluate various conjugatedgold nanospheres. The images acquired at 45 kVp and 80 kVp were observedon the ImageJ software and a mean of five ROI (region of interest)values is obtained for the attenuation values in Hounsfield Unit (HU).The solution of GNS-DO3A-Gd3+ decorated with AS1411 yielded a highermagnitude of X-ray attenuation intensities and Hounsfield units permilligram milliliter (slope) values in comparison to equivalent amountsof the industry standard (Iopamidol) or citrate-capped goldnanoparticles (FIGS. 10A & 10B). In general, the attenuation intensitiesincrease with gold nanoparticle concentration and therefore theGNP-Gd(III)-DO3A-SH-Oligo probes can be used as an contrast agent for CTimaging modality.

Clonogenic Assay of Lung Cancer Cells

Lung cancer cells (A549) were treated with gold nanoparticles (GNP), 100nM gold nanoparticle-control aptamer conjugates (GNP-CRO) and 100 nMgold-nanoparticle-AS1411 conjugates (GNP-AS1411). After 36 hours, thecells were exposed to 100 cGy X-ray radiation. The cells were furtherincubated for 7 days and fixed with 4% formalin and stained with 0.4%crystal violet solution. Colonies were de-stained with 10% acetic acidand absorbance was measured at 570 nm. FIG. 11A shows treated lungcancer cells before exposure to radiation. FIG. 11B shows treated lungcancer cells after exposure to radiation. FIG. 11C is a graphillustrating the percent change in absorbance (after de-staining with10% acetic acid) of the cells before and after exposure to radiation.The results indicate that the AS1411 conjugates enhance the effects ofradiation on lung cancer cells.

Concentration Study in Breast Cancer Cells

Triple negative breast cancer cells (MDA-MB-231) were treated withvarying concentrations of gold nanoparticles (GNP), goldnanoparticle-control oligonucleotide conjugates (GNP-CRO) andgold-nanoparticle-AS1411 conjugates (GNP-AS1411) for 72 hours. X-rayradiation was then applied to the cells. The Log₁₀ Survival Fraction ofthe cells after treatment and application of varying doses of X-rayradiation (cGy) was plotted. FIG. 13A shows the cancer cell survivalfraction at 1.38 μg/mL. FIG. 13B shows the cancer cell survival fractionat 2.76 μg/mL. FIG. 13C shows the cancer cell survival fraction at 5.5μg/mL. The results indicate that GNP-AS1411 conjugates become moreeffective at killing cancer cells at increasing concentrations.

Clonogenic Survival Assay of Breast Cancer Cells

Breast cancer cells (MDA-MB-231) were treated with gold nanoparticles(GNP), gold nanoparticle-control oligonucleotide conjugates (GNP-CRO)and gold-nanoparticle-AS1411 conjugates (GNP-AS1411) for 72 hours. Thecells were washed in PBS and radiated at varying doses of γ-rays using aGammaCell-40 irradiator. The cells were trypsinized, counted and platedin six well plates for an additional 10 days. After incubation, thecells were fixed with methanol and stained with 2% crystal violet. Thecells were counted and plotted for survival fraction as a function ofradiation dose after exposure to γ-radiation. The results of theradiation treatment are shown in FIG. 14A. The Log₁₀ Survival Fractionof the cells versus the X-ray radiation dose in cGy is shown in FIG.14B. The results indicate that the AS1411 conjugates enhance the effectsof radiation on breast cancer cells.

Selective Retention of Gold Nanoparticle-Gadolinium-Oligomer Conjugatesin Malignant Cells

Human immortalized breast epithelial cells (MCF10A) (non-malignant) andtriple negative breast cancer cells (MDA-MB-231) were incubated with offluorescent-labeled gold nanoparticle-gadolinium-oligomer conjugates for4 and 96 hours. The cells were incubated with 50 nM GNP—Gd-CRO-Cy5(control oligomer) or 50 nM GNP-Gd-AS1411-Cy5 using DO3A(1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane) as the ligandto bind Gd and connect it to the GNP surface through a thiol linker.After incubation, the cells were harvested and washed with PBS (2×),counted using a TC-10 Cell Counter (Bio-Rad), plated in a 4 chamberslide (Bio-Tek) at 1,000 cells/well and incubated for an additional 24hours. The cells were stained with nuclear stain DAPI after incubationand the complete DMEM media was replaced with DMEM without phenol red.Images were acquired using a NIKON® confocal microscope. FIG. 15A showsuptake in non-malignant breast cells. FIG. 15B shows uptake andselective retention of GNP-Gd-AS1411-Cy5 in malignant breast cancercells at the 96 hours timepoint. The fluorescent-labeled GNP conjugatesappear red and the nuclear stain appears blue.

Human normal airway epithelial cells (HPLD-1) (non-malignant) and lungadenocarcinoma cells (A549) were incubated with of fluorescent-labeledgold nanoparticle-gadolinium-oligomer conjugates for 4 and 96 hours. Thecells were incubated with 50 nM GNP—Gd-CRO-Cy5 (control oligomer) or 50nM GNP-Gd-AS1411-Cy5 using DO3A as the ligand to bind Gd and connect itto the GNP surface through a thiol linker. After incubation, the cellswere harvested and washed with PBS (2×), counted using a TC-10 CellCounter (Bio-Rad), plated in a 4 chamber slide (Bio-Tek) at 1,000cells/well and incubated for an additional 24 hours. The cells werestained with nuclear stain DAPI after incubation and the complete DMEMmedia was replaced with DMEM without phenol red. Images were acquiredusing a NIKON® confocal microscope. FIG. 16A shows uptake innon-malignant lung cells. FIG. 16B shows uptake and selective retentionof GNP-Gd-AS1411-Cy5 in malignant lung cancer cells at the 96 hourstimepoint. The fluorescent-labeled GNP conjugates appear red and thenuclear stain appears blue.

The 4 hour images indicate that the uptake of aptamer conjugates inhealthy and malignant cells is similar. The 96 hour images indicate thatthe AS1411 conjugates are only retained in malignant cells.

Gold Nanoparticle-Gadolinium-Oligomer Conjugates as MRI Contrast Agents

Gold nanoparticle-gadolinium-oligomer conjugates were studied as MRIcontrast agents in vitro and in cells using a Bruker BioSpec 94/30 USR9.4T MRI scanner. The change in reflectivity was measured using water tonormalize the signals.

In a first experiment, the correlation between the concentration of thegold nanoparticle-oligomer conjugates and the improvement in thereflectivity due to the gadolinium (III) contrast agent was studied. Thegold nanoparticle-oligomer conjugates included gold nanoparticlesconjugated to AS1411 (5′-d(GGTGGTGGTGGTTGTGGTGGTGGTGG)-3) (GNP-AS1411),gold nanoparticles conjugated to a control oligomer(5′-d(CCTCCTCCTCCTTCTCCTCCTCCTCC)-3) (GNP-CRO) and gold nanoparticlesconjugated to a control oligonucleotide (5′d(TTTT)-3) (GNP-CTR). DO3Aligand was used to bind Gd(III) and connected it to the GNP surfacethrough a thiol linker. GNP-AS1411, GNP-CRO and GNP-CTR were used ascontrols to compare the relaxivity of GNP-Gd-AS1411, GNP—Gd—CRO andGNP—Gd-CTR. The relaxivity was measured at 75 nM, 300 nM and 1200 nM GNPconcentrations. Table 4 shows the change in relaxivity afteradministration of the Gd conjugates:

TABLE 4 Change in relaxivity GNP-Gd-CTR GNP-Gd-CRO GNP-Gd-AS1411  75 nM17% 15% 19%  300 nM 33% 25% 44% 1200 nM 59% 58% 64%

These results are shown graphically in FIG. 17A. The results show nosignificant change in relaxivity between particles for the sameconcentration.

In a second experiment, the correlation between the concentration of thegold nanoparticle-oligomer conjugates in cells and the improvement inthe reflectivity due to the gadolinium (III) contrast agent was studied.30,000,000 cells were treated with GNP-AS1411, GNP-CRO, GNP-CTR,GNP-Gd-AS1411, GNP—Gd—CRO or GNP—Gd-CTR for 48 hours or 96 hours. Therelaxivity was measured at 75 nM, 300 nM and 1200 nM GNP concentrations.Table 5 shows the change in relaxivity after the 48 hour treatment:

TABLE 5 Change in relaxivity after 48 hour treatment 48 hour treatmentGNP-Gd-CTR GNP-Gd-CRO GNP-Gd-AS1411  75 nM 5.5% 5% 6%  300 nM 5.7% 8% 9%1200 nM  22% 19%  37% 

Table 6 shows the change in relaxivity after the 96 hour treatment:

TABLE 6 Change in relaxivity after 96 hour treatment 96 hour treatmentGNP-Gd-CTR GNP-Gd-CRO GNP-Gd-AS1411  75 nM 2.2% 3.5% 3.6%  300 nM 4.7%7.1% 8.1% 1200 nM 15.3% 17.2% 36.8%

The 48 hour treatment results are shown graphically in FIG. 17B, and the96 hour treatment results are shown graphically in FIG. 17C.GNP-AS1411-Gd demonstrated a significant increase in relaxivity at 1200nM after 48 hours and 96 hours of treatment.

Comparison of Gold Nanoparticle-Oligomer Conjugates toCommercially-Available MRI Contrast Agent

The MRI contrast enhancement properties of gold nanoparticle-oligomerconjugates were compared to MULTIHANCE® (Bracco), acommercially-available MRI contrast agent. The percent contrastenhancement was measured in breast cancer cells (MDA-MB-231). The goldnanoparticle-oligomer conjugates included gold nanoparticles conjugatedto AS1411 (5′-d(GGTGGTGGTGGTTGTGGTGGTGGTGG)-3) (GNP DOTA AS1411), goldnanoparticles conjugated to a control oligomer(5′-d(CCTCCTCCTCCTTCTCCTCCTCCTCC)-3) (GNP DOTA CRO) and goldnanoparticles conjugated to a control oligonucleotide (5′d(TTTT)-3) (GNPDOTA CTR). DOTA(1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane) wasused as the ligand. The MULTIHANCE® cells were treated for 24 hours, 48hours, 72 hours and 96 hours, while the gold nanoparticle-oligomerconjugate cells were treated for 48 hours and 96 hours. The results areshown in FIG. 18. GNP DOTA AS1411 demonstrated a contrast enhancementapproximately three times greater than MULTIHANCE®.

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1. A composition, comprising an anti-nucleolin agent and gadoliniumconjugated to nanoparticles.
 2. The composition of claim 1, wherein thenanoparticles comprise at least one member selected from the groupconsisting of metals, elements and oxides.
 3. (canceled)
 4. Thecomposition of claim 1, wherein the anti-nucleolin agent is selectedfrom the group consisting of an anti-nucleolin oligonucleotide, anantibody, a nucleolin targeting protein and a guanosine-richoligonucleotide (GRO). 5-8. (canceled)
 9. The composition of claim 1further comprising a cyanine dye.
 10. The composition of claim 1,wherein the nanoparticles have an average diameter of 1 to 50 nm. 11.The composition of claim 1, wherein the nanoparticles have an averagediameter of 1 to 20 nm.
 12. The composition of claim 1, wherein thenanoparticles comprise at least one member selected from the groupconsisting of gold, platinum, iridium and palladium.
 13. The compositionof claim 1, wherein the gadolinium is present as a complex of gadolinium(III).
 14. The composition of claim 1, wherein the gadolinium is presentas 10-(2-sulfanylethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (H3-DO3A-SH) coordinated to the gadolinium ion (Gd-DO3A-SH). 15-17.(canceled)
 18. A method of treating cancer, comprising administering aneffective amount of a pharmaceutical composition comprising ananti-nucleolin agent conjugated to nanoparticles, to a patient in needthereof, followed by radiation therapy.
 19. The method of treatingcancer of claim 18, wherein the cancer is selected from the groupconsisting of melanoma, lymphoma, plasmocytoma, sarcoma, glioma,thymoma, leukemia, breast cancer, prostate cancer, colon cancer, livercancer, esophageal cancer, brain cancer, lung cancer, ovary cancer,cervical cancer and hepatoma.
 20. (canceled)
 21. The method of treatingcancer of claim 18, further comprising administering a second cancertreatment selected from the group consisting of vinorelbine, mitomycin,camptothecin, cyclophosphamide, methotrexate, tamoxifen citrate,5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel,docetaxel, vinblastine, imatinib mesylate, anthracycline, letrozole,arsenic trioxide, anastrozole, triptorelin pamoate, ozogamicin,irinotecan hydrochloride, BCG live, leuprolide acetate implant(VIADUR®), bexarotene, exemestane, topotecan hydrochloride, gemcitabineHCL, daunorubicin hydrochloride, toremifene citrate (FARESTON®),carboplatin, cisplatin, oxaliplatin, trastuzumab, lapatinib, getitinib,cetuximab, panitumumab, temsirolimus, everolimus, vandetanib,vemurafenib, crizotinib, vorinostat, bevacizumab, hyperthermia, genetherapy and photodynamic therapy. 22-25. (canceled)
 26. A method ofimaging cancer by MRI and/or X-ray imaging techniques in vivo,comprising: administering a composition comprising an anti-nucleolinagent and gadolinium conjugated to nanoparticles to a subject; andforming an image of the imaging agent present in the subject by MRIand/or an X-ray imaging technique.
 27. The method of imaging cancer ofclaim 26, wherein the administering occurs more than 24 hours before theforming.
 28. The method of imaging cancer of claim 26, wherein theadministering occurs more than 48 hours before the forming.
 29. Themethod of imaging cancer of claim 26, wherein the cancer is lung cancer.30. The method of imaging cancer of claim 26, wherein the method iscapable of identifying malignant lesions in the lung that are less than4 mm.
 31. The composition of claim 1, wherein the anti-nucleolin agentcomprises AS1411.
 32. The method of treating cancer of claim 18, whereinthe anti-nucleolin agent comprises AS1411.
 33. The method of imagingcancer of claim 26, wherein the anti-nucleolin agent comprises AS1411.